An important requirement for platelet function is speed. Theoretical calculations suggest an activation time as short as several milliseconds. Recent research indicates that physical changes may begin by 200 to 300 milliseconds, and that biochemical events, such as phospholipid hydrolysis, can occur within 5 seconds of an inducing stimulus. However, kinetics and inter-relationships are not clear. One of the major problems in understanding these fundamental early reactions has been the inability to react platelets for very short but precisely-controlled times. A quenched flow approach has been developed for this purpose. Reaction characteristics between about 20 milliseconds to minutes or longer can now be studied. An automated, resistive-particle counter has been used to investigate the kinetics of aggregation and volume changes; the former has an activation time of about 1 second, the latter 0.3. The proposed research will use the quenched-flow system to investigate the nature, sequence and factors regulating the early structural and biochemical events leading to platelet aggregation. Surface changes in platelet structure will be examined by the technique of spray freezing, followed by scanning electron microscopy. Freeze-fracture together with the new technique of deep-etching and transmission electron microscopy will be employed to analyze potential membrane and internal structural rearrangements. Biochemical studies will concentrate on the onset time, speed of formation, and nature of metabolites resulting from both phospholipase A2 and C action. These factors will be analyzed in relation to potential changes in adenine nucleotide and cyclic AMP concentrations, levels of phosphorylation of membrane proteins, and the sequence of morphological events. A resistive-particle counter will be used to relate the biochemical and structural changes to the kinetics of the initial "shape change" and subsequent platelet aggregation.